Microbial contamination, such as Gram positive bacteria, Gram negative bacteria, yeast, and fungi may cause severe illness and even death in humans. When people become infected with gram negative bacteria, the bacteria may produce fever-inducing bacterial endotoxins or endotoxin molecules. Endotoxin molecules may be a source of contamination even when no microbes are present. Endotoxin molecules can be dangerous, lead to toxic shock, or even be deadly to humans.
Manufacturers in certain industries, especially the pharmaceutical, medical device and food industries, must meet certain standards to make sure their products do not contain microbial or endotoxin contamination. These industries require frequent, accurate, and sensitive testing for the existence of endotoxins to meet various safety standards, such as those set by the United States Food and Drug Administration, or the Environmental Protection Agency.
Currently, a variety of assays have been developed to detect the presence of endotoxin in or on the sample being tested using hemocyte lysates from horseshoe crabs. Clotting will occur when the hemocyte lysate is exposed to the endotoxin. Hemocyte lysate is amoebocyte lysate produced from the hemolymph of various horseshoe crab species, including the Limulus, Tachypleus, and Carcinoscorpius species. A commonly used amoebocyte lysate, produced from the hemolymph of the Limulus species, is referred to as Limulus amoebocyte lysate (“LAL”).
Routine assays that use LAL include, gel clot assays, end point turbidimetric assays, kinetic turbidimetric assays, endpoint chromogenic assays, and kinetic chromogenic assays. More information on these assays and the standards used may be found in United States Pharmacopeia (“USP”) Chapter 85 “Bacterial Endotoxins Test” (“BET”), Japanese Pharmacopeia 4.01 “Bacterial Endotoxin Test”, European Pharmacopoeia 2.6.14 “Bacterial Endotoxins”, and other equivalent national Pharmacopeias. Additional internationally harmonized pharmacopeia information can be found in ICH Q4B Annex 14 “Bacterial Endotoxin Test General Chapter”. For endotoxin testing in medical devices, information can be found in USP Chapter 161 “Transfusion and Infusion Assemblies and Similar Medical Devices” and ANSI/AAMI ST72 “Bacterial endotoxins—Test methods, routine monitoring, and alternatives to batch testing”.
Typical chromogenic and turbidometric BET assays are run in well plates, with measurements of absorbance being conducted by plate readers designed to run 24, 96 and 384 well plates. In all these cases, the kinetic assay requires measurement of time required to reach a specified onset optical density (absorbance). Therefore, absorbance values are measured at periodic intervals for all samples and standards being tested in a single plate. As the reaction progresses, the optical density (“OD”) increases with time, but it is impossible to expect that an actual measurement be equal to the exact specified onset optical density. Data analysis is required for data on consecutive measurements, in order to calculate the time at which the optical density of the sample reached and passed the specified onset value. Such analysis typically involves either a simple linear interpolation between two consecutive time-stamped data points with measured values less than and greater than the specified onset value, or linear or non-linear curve fitting over a larger set of data points in the neighborhood of the specified onset value. The time required to reach onset OD is inversely correlated with endotoxin concentrations, so that testing for low levels of endotoxin necessarily requires longer assay times. This is typically an hour or more for measuring 0.005 EU/mL distinguished from a blank (negative control), depending on the manufacturer and type of the LAL reagent.
Turbidimetric assays usually require longer times than the chromogenic assays using comparable LAL reagent in conjunction with a chromophoric substrate. The chromogenic assays, which incorporate a chromogenic substrate with an absorbance peak at 405 nm, may be performed either in an endpoint mode or in a kinetic mode. In chromogenic assays, the time dependence of the optical density measured at 405 nm shows a substantial initial period during which the OD remains close to zero, often called “initial lag time”. The initial lag time is followed by a period in which OD starts to rise, or “initial slope”. The initial lag time becomes shorter as the endotoxin concentration increases, while the initial slope of the OD response increases with endotoxin concentration. The endpoint mode correlates endotoxin levels with the OD at 405 nm achieved at a specified time. The kinetic mode correlates endotoxin levels with the time required to achieve a specified onset OD at 405 nm, typically defined as OD=0.2. Both the endpoint assay and kinetic assay have long run times, typically greater than 60 minutes, in order to get the resolution between a blank control and desired lower detection limits of 0.005 EU/mL or 0.001 EU/mL.
In cases where assay results need to be obtained quickly, it is therefore desirable to have methods of data analysis that correlate reliably with endotoxin concentration at earlier times in the assay. As such, several methods have been developed to estimate endotoxin concentration at earlier times in the assay.
One method is a dynamic light scattering method that obtains results from a turbidimetric assay quicker than the currently performed static light scattering method. Another method uses an apparatus that uses light scattering measurements for turbidimetric assay in conjunction with a time function difference method and a multi-series difference method to correlate assay times with endotoxin concentration. Another method uses a correlation of endotoxin concentration with initial rate of increase in scattered light detected in a turbidimetric assay. Although all these methods may reduce the time required to obtain the endotoxin concentration of a given sample, they all rely on correlation or linear methods of forecasting. Nor do these methods allow the user to correlate the data to compendia requirements or standards. Moreover, none of these methods are capable of alerting the user to anomalies in the measurement.